The infrequently dividing or so-called slow-cycling nature of cells within tissues has been linked to stemness in several adult tissues including mouse skin. Slow-cycling cells defined as H2B-GFP or DNA label retaining cells (LRCs) are thought to reside at the top of the lineage hierarchy, as stem cells, giving rise to actively-cyclig cells (non-LRCs), which may function as transit-amplifying (TA) short-lived progenitor cells. Despite the crucial importance of the epidermis for essential body functions and for skin regeneration therapy, molecular characterization of stem and TA cells within the basal layer of the inter-follicular epidermis has not been achieved. Consequently, we lack the specific markers to accurately define the dynamic stem cell behavior within this tissue and to understand the regulatory factors maintaining stemness. In our preliminary study, we have isolated LRCs and non-LRCs from the basal layer of the epidermis and analyzed the molecular signature of these cells by microarray. Here we propose to characterize the expression pattern of several putative epidermal stem and TA cell markers identified in our microarray, including one novel cell surface marker for basal layer LRCs. We will analyze the lineage potential of subpopulations of basal layer cells by three functional assays: colony formation activity, regeneration potential in cell transplantation, and lineage tracing. These data will reveal long-term stem cell potential and lineage hierarchy of epidermal basal cell subpopulations in vitro and in vivo and will provide new tools for studying stem cell regulation in the epidermis. Data obtained from our proposed experiments will have global relevance for stem cell-based therapy of human skin injury or skin replacement, where slow-cycling cells are thought to play central roles.